Elastin like polypeptide and hydrophobin fusion proteins expressing transgenic tobacco plants consisted of intracellular protein bodies.
Production of recombinant proteins is found to be easy in the case of plants as the process here is efficient and cost effective. The methods by which recombinant proteins are expressed in plants are transformation of plant genome and Agrobacterium based transient expression. There are two confines for the plants to be used as bioreactors. These limitations are absence of proficient purification procedures and reduced build-up of recombinant proteins. Elastin like polypeptide or ELP and hydrophobin-1 are the fusion molecules which have the capacity to enhance the concentration of recombinant proteins and stimulate the formation of protein bodies in the plant leaves through transient expression assay. The current scientific study involves the large accumulation of recombinant protein by ELP and HFB1 fusion tags accompanied by the formation of protein bodies in the stable transgenic tobacco plant (Nicotiana tobacum). The two cultivars of tobacco that were used in this study for the recombinant protein expression were 164 and 81V9.
Previously published plant expression vectors were utilized for stable transformation of tobacco. The transformation experiment was conducted to study the impact of ELP and HFB1 tags on the green fluorescent protein (GFP) accumulation and in the stimulation of protein body formation in the transgenic tobacco plant. The three constructs that were used for transgenic tobacco plants were GFP, GFP-ELP and GFP-HFB1 and they expressed the respective fusion proteins targeted at Endoplasmic reticulum driven by cauliflower mosaic virus 35S promoter. The fusion proteins were expressed also under the control of nopaline synthase terminator (nos), expression vector in plants like pCaMterX and translational enhancer tCUP.
The variation in the accumulation levels of recombinant protein in different transgenic lines is described by the insertion of the transgene at various positions of the genome, shutdown of expression of transgene by certain silencing agents and copy number of the transgene. The tobacco plants that were expressing free GFP did not accumulate much of the recombinant protein compared to those that expressed fusion proteins GFP-ELP and GFP-HFB1.
Statistical validation was done for confirming that the accumulation levels of the recombinant protein was enhanced by the fusion tags. The normality test conducted for first generation transgenic plants on recombinant protein quantification has come out to be a failure probably due to the small population of the first generation. Later, Kruskal-Wallis test was performed to estimate the statistical differences existing between the four transgenic lines used in the experiment. This test was followed by Wilcoxon-Mann-Whitney test conducted to compare the values in four transgenic lines. The results revealed that HFB1 and ELP tags significantly improved the accumulation of GFP in stable transgenic plants compared to the plants expressing GFP without fusion tags. Again HFB1 was observed to be significantly increasing the accumulation of the GFP than ELP. No significant difference was observed in the expression of GFP-HFB1 between the 164 and 81V9 cultivars of tobacco.
The formation of plant bodies was investigated by observing the GFP, GFP-ELP and GFP-HFB1 proteins targeted to ER using Confocal Laser scanning microscopy. The microscope was used to observe the leaves in number of different transgenic plants having old leaf, medium sized and young leaf. The intracellular fluorescence network pattern of ER was observed in the transgenic lines expressing GFP. Apart from the fluorescence expression in these plants, small spherical particles of size ranging from 0.2 to 0.5 um were found that were same as the protein bodies observed in the transient expression assays done with the leaves of Nicotiana benthamiana.
The leaf cells of transgenic plants expressing GFP-ELP and GFP-HFB1, showed protein bodies distributed inside. These PBs were found in similar numbers in both the cultivars and their diameters ranged from 0.5 um to 2 um. Most of the protein bodies had the size range of 0.5 um to 1 um. The large sized protein bodies were found to be present even if the accumulation levels of the recombinant protein were higher. It was interesting to observe that the protein bodies are found in the guard cells of the transgenic plants irrespective of the tobacco cultivars and the type of construct expressed in it. The guard cells were having more PBs than the epidermal cells that are surrounding them in the 81V9 cultivar transgenic plants expressing GFP-HFB1.
The accumulation of recombinant protein was found to be related to the formation of protein bodies. It is observed that when the recombinant protein accumulation is higher, the protein bodies are formed. The transgenic plants with low levels of recombinant protein accumulation had no PBs present in them while those with increased levels had PBs in all the cells. Therefore, it is concluded that a limit of 0.2 percent of GFP expression is necessary for the PBs to appear in the transgenic plant leaves in any of the cultivars.
Another feature observed in these transgenic plants which had low accumulation levels of GFP is the presence of post transcriptional gene silencing mechanism. The accumulation of the recombinant proteins in transgenic tobacco plants was also found to be influenced by post transcriptional gene silencing.
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